ROLE OF CAMP-DEPENDENT PROTEIN KINASE IN GROWTH CONTROL

我们的长期目标是通过深入研究PKA调节(R)和催化(C)亚单位的结构、功能和表达来了解cAMP依赖的蛋白激酶(PKA)对细胞生长的调控。PKA的两种亚型PKA-I和PKA-II共享一个共同的催化(C)亚基，但分别含有不同的调节(R)亚基RI和RII。已知PKA-I及其调节亚基RIA在人类癌细胞系、原发肿瘤和转化后的细胞中的表达增加。我们的研究发现：(1)反义策略的使用：利用RIA反义寡核苷酸或反义RIA基因的过表达来证明序列特异性地抑制RIA基因的表达，并在体内抑制肿瘤的生长。RIA反义缺失导致RIIb和PKA-IIb全酶迅速升高。脉冲追逐实验表明，RIIb蛋白在反义处理细胞中的半衰期增加了3-6倍。因此，全酶复合体中的RIIb是稳定的，表现出增加的半衰期。通过这种生化适应，在反义处理的癌细胞中，PKA-I/PKA-II的比例与正常细胞相似。(2)重组基因技术：逆转录病毒载体介导的PKA野生型和点突变的RIA、RIIA、RIIB和C亚基的过表达，展示了R亚基异构体在细胞生长、分化和逆转中的独特作用；(3)细胞外PKA的发现(ECPKA)：在各种癌细胞的条件培养液中和癌症患者的血清中分泌PKA的自由催化(C)亚基；与正常血清相比，ECPKA表达上调10倍。重要的是，本研究提供了通过改变细胞内PKA-I和PKA-II的表达来调控ECPKA表达的方法。也就是说，(1)在表达载体中过表达RIA，上调细胞内PKA-I的表达，可以显著上调ECPKA的表达；(2)过表达RIIb，下调细胞内的PKA-I，并逆转转化的表型，下调ECPKA的表达；(3)阻止肉豆蔻酸化的Ca基因突变，允许细胞内PKA上调，但阻止ECPKA的增加，这表明ECPKA的表达需要钙的N端肉豆蔻花柱基团。进一步研究表明，与激素非依赖性乳腺癌相比，维持激素依赖性(一种正常细胞特性)的癌细胞，如激素依赖型乳腺癌，ECPKA上调减少。这些观察表明，ECPKA的这种表达现象可以为癌症的诊断、预后和激素依赖性检测(乳腺癌)提供创新的方法。

Our long term goal is to understand the regulation of cell growth by cAMP-dependent protein kinase (PKA) by an in-depth investigation of the structure, function and expression of the regulatory (R) and catalytic (C) subunits of PKA. Two isoforms of PKA, PKA-I and PKA-II, share a common catalytic (C) subunit but contain distinct regulatory (R) subunits, RI and RII, respectively. It is known that expression of PKA-I and its regulatory subunit, RIa is increased in human cancer cell lines, in primary tumors, and in cells after transformation. Our studies revealed the following: (1) Use of antisense strategy: RIa antisense oligodeoxynucleotide or antisense RIa gene overexpression is used to demonstrate the sequence-specific inhibition of RIa gene expression and in vivo tumor growth inhibition. The loss of RIa by the antisense resulted in rapid increase in RIIb and PKA-IIb holoenzyme. Pulse-chase experiments demonstrated that RIIb protein increased its half-life 3-6 fold in antisense treated cells. Thus, RIIb in the holoenzyme complex is stabilized, exhibiting an increased half-life. Through this biochemical adaptation, in the antisense-treated cancer cells, the ratio of PKA-I to PKA-II changes to that similar to that of normal cells.; (2) Recombinant gene technology: Retroviral-vector mediated overexpression of wild type and point-mutated RIa, RIIa, RIIb and C subunits of PKA to demonstrate distinctive roles of the R subunit isoforms in cell growth, differentiation, and reverse transformation; and (3) Discovery of extracellular PKA (ECPKA):the free catalytic (C) subunit of PKA is excreted in conditioned medium of various cancer cells, and in serum of cancer patients; the ECPKA expression was upregulated 10-fold as compared with normal serum. Importantly, this study provided the means of modulating the ECPKA expression by changing expression of the intracellular PKA-I and PKA-II. Namely, (1) overexpression of RIa in expression vector, which upregulates intracellular PKA-I, can markedly upregulate ECPKA expression; (2) overexpression of RIIb, which downregulates PKA-I in the cell and reverts the transformed phenotype, downregulates ECPKA; and (3) A mutation in the Ca gene that prevents myristylation allows the intracellular PKA upregulation but blocks the ECPKA increase, suggesting that the N-terminal myristyl group of Ca is required for ECPKA expression. It was further shown that ECPKA upregulation is reduced in cancer cells maintaining hormone-dependency (a normal cell property), such as hormone-dependent breast cancer as compared to the hormone-independent breast cancer. These observations indicate that this phenomenon of ECPKA expression can provide innovative approach to cancer diagnosis, prognosis, and hormone-dependence detection (breast cancer).